Endophytic microbial biostimulant

Abstract

The present invention relates to a composition comprising:-an isolated endophyte wherein the endophyte is a strain of Bacillus xylaniformis, and-an adjuvant wherein the strain of Bacillus xylaniformis has accession number DSM 34353.

Description

Field of the Invention

[0001] The present invention relates to novel isolated plant microbiome strains, particularly endophytes, plants infected with such strains, and related methods. Specifically, the isolated endophytes can be used in compositions for use as biofertilizers and / or biostimulants or for bioprotection in plants. The compositions may further comprise adjuvants. Background of the Invention

[0003] In many cases, the growth and / or productivity of crops are subject to the limitation of the amount of nitrogen that can be used or absorbed by plants. In order to overcome this limitation, exogenous nitrogen is added to the soil before or after sowing. Nitrogenous fertilizers can have organic (for example, urea, amino acid, manure, horn crumbs) or mineral (for example ammonium nitrate, ammonium sulfate, potassium nitrate) source in nature. The common point that all these artificial nitrogenous fertilizers have is that their production, transportation and / or application are all energy intensive, and therefore result in positive CO balance. Especially, ammonium nitrate and urea, the most widely used nitrogenous fertilizers in the world so far, have large CO footprint (footprint), because their common precursor ammonia (NH ) is derived from Haber-Bosch process (Haber-Bosch process). The method uses atmospheric nitrogen (N ) and hydrogen (H ) to synthesize ammonia (NH ) under high temperature and high pressure. Therefore, the method aggravates climate change and is unfavorable to the environment.

[0004] An alternative and more sustainable way to provide plants with at least some of the nitrogen they need is to use biofertilizers. Biofertilizers consist of living microorganisms that can reduce atmospheric nitrogen and thus make it available to plants in the form of ammonium / ammonia or organically bound nitrogen. These organisms must be applied in a metabolically active form to the seeds, roots, or, more rarely, leaves.

[0005] In the past and current use of these biofertilizers, nitrogen-fixing microorganisms are applied to the surface, leaf or soil of seed. Due to this fact, biological exposure is exposed to the high stress from abiotic factors such as temperature, drought, pH and from the washing away of rainwater and high humidity. The effectiveness and efficiency of biofertilizers are reduced. Therefore, a large amount of biofertilizers are needed so that it effectively provides the biological protection for plant and / or increases the productive rate of crop. This has increased agricultural costs.

[0006] Furthermore, biological factors such as high competition with already dominant plant or soil microbiota or the presence of antibiotic active substances also reduce the survival rate of exogenously added nitrogen-fixing microorganisms on the respective surfaces. This also reduces the effectiveness of currently available biofertilizers.

[0007] Accordingly, there is a need in the art for means to better utilize these endophytes as biofertilizers and / or biostimulants to improve sustainable agriculture and the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The figures are pictures of maize growth (no treatment) after 6 to 8 weeks under different nitrogen fertilizations (0 kg N / ha, 21 kg N / ha, 42 kg N / ha). Nutrient-poor Oxisol soil was used as the test soil (substrate).

[0010] Figure 2 The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2. Pictures of corn growing in a mixture of 1% glycerol (applied only to the leaves).

[0011] Figure 3 The results show that the growth of Paenibacillus xylans with and without Paenibacillus xylans DSM 34353 (which is Graph of shoot dry matter of maize applied in a mixture of .

[0012] Figure 4 The results show that the growth of Paenibacillus xylans with and without Paenibacillus xylans DSM 34353 (which is Graph of root dry matter of maize applied in a mixture of .

[0013] Description of the Invention

[0014] The present invention attempts to solve the above problems by providing novel isolated endophytes from Paenibacillus xylanexedens that can become effective and efficient biostimulants and / or biofertilizers. Due to their unique genetic equipment and / or in the presence of specific adjuvants, these newly isolated endophytes can allow and especially enhance the penetration of these endophytes into plants or parts thereof with which the endophytes are in contact. In particular, in the presence of appropriate adjuvants, the newly isolated Paenibacillus xylanexedens strains are able to penetrate and proliferate in plant tissues or seeds. In this way, (abiotic) stress can be reduced and the viability or effectiveness of the organisms in the plant can be significantly increased. Within the plant or seed or any part thereof, the active Paenibacillus xylanexedens strain is in a regulated steady state, which increases both the efficacy and viability of the nitrogen-fixing endophyte.

[0015] According to one aspect of the present invention there is provided a substantially purified or isolated endophyte, wherein the endophyte is a strain of Paenibacillus xylanicum, wherein the strain of Paenibacillus xylanicum has accession number DSM 34353 and which provides a bioprotective and / or biostimulant phenotype to a plant into which it is introduced.

[0016] According to any aspect of the present invention, the newly isolated endophyte Paenibacillus xylanicum having accession number DSM 34353 solves the problem of low stability of microbial nitrogen fixers because it is an endophyte with both unprecedented high nitrogen fixation capacity and the ability to penetrate into the plant endosphere, two abilities that most other existing endophytes used in agriculture do not possess.

[0017] Endophytes according to any aspect of the present invention were isolated from the internal plant tissues of copper flower (Minuartia vernasubsp.hercynica) and were typed and sequenced as Paenubacillus xylanus species. Greenhouse experiments demonstrated that Paenubacillus xylanus was able to provide a significant amount of enzymatically fixed nitrogen to maize (Zea mays, variant LG31.224) both in the seed and foliage applications. Thus, maize inoculated or sprayed with Paenubacillus xylanus showed growth that was identical or even improved to fully fertilized maize (42 kg / ha) when fertilized with only half a synthetic nitrogen fertilizer (21 kg / ha). In contrast, significantly reduced growth was observed in maize not inoculated with Paenubacillus xylanus at 21 kgN / ha.

[0018] As used herein, the term "endophyte" is an endosymbiont, which refers to a bacterial or fungal strain that lives within a plant for at least part of its life cycle without causing overt disease. In particular, the bacterium or fungus is closely associated with the plant, wherein the term 'closely associated' refers to the bacterium or fungus living on, in, or near the plant. For example, it may be endophytic and live within the internal tissues of the plant, or it may be epiphytic and grow externally on the plant. There are many different endophytes that have been discovered. However, only a few have been used commercially as endophyte inoculants for agriculture, such as arbuscular mycorrhizae, rhizobia, and Azospirillium. Clavicipitaceous fungi are also endophytes used in agriculture. An endophyte according to any aspect of the invention is a strain of Paenibacillus xylanus with accession number DSM 34353.

[0019] As used herein, the term "substantially purified" refers to an endophyte that is free of other organisms. The term includes, for example, endophytes in axenic culture. In particular, the endophyte is at least about 90% pure, more particularly at least about 95% pure, and even more particularly at least about 98%, 99%, or 99.5% pure.

[0020] As used herein, the term 'isolated' refers to an endophyte according to any aspect of the present invention that has been removed from its original environment (e.g., the natural environment if it occurs naturally). For example, a naturally occurring endophyte present in a living plant is not isolated, but the same endophyte separated from some or all coexisting materials in the natural system is isolated. In particular, the endophyte isolated according to any aspect of the present invention may be a pure culture of a single strain and the single strain was submitted to the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig, Germany on August 11, 2022, and has accession number DSM 34353. Particles for preserving and modifying cells are available from the prior art, for example Sambrook / Fritsch / Maniatis (1989).

[0021] As used herein, the term "bioprotectant and / or biostimulant" may refer to an endophyte according to any aspect of the present invention having genetic and / or metabolic properties that result in beneficial phenotypes in plants containing or otherwise associated with the endophyte. Such beneficial properties or phenotypes resulting from the presence of the endophyte in a plant include increased resistance to pests and / or diseases, improved tolerance to water and / or nutrient stresses, enhanced biotic stress tolerance, enhanced drought tolerance, enhanced water use efficiency, reduced toxicity, and increased vigor in the plant to which the endophyte is associated, compared to plants not associated with the endophyte according to any aspect of the present invention or with an endophyte, such as a standard toxic (ST) endophyte. In particular, the bioprotectant and / or biostimulant phenotype according to any aspect of the present invention includes nitrogen fixation in plants into which the endophyte has been introduced.

[0022] Pests and / or diseases may include, but are not limited to, fungal and / or bacterial pathogens, particularly fungi. In one example, an endophyte may cause the production of a bioprotective compound in the plant with which it is associated.

[0023] As used herein, the term 'bioprotectant compound' refers to a compound that provides or helps provide biological protection to the plant with which it is associated against pests and / or diseases, such as bacterial and / or fungal pathogens. Bioprotectant compounds may also be referred to as 'biocidal compounds'.

[0024] As used herein, the term 'biostimulant' refers to any substance or microorganism applied to plants with the purpose of enhancing nutrient efficiency, abiotic stress tolerance, and / or crop quality traits, regardless of their nutrient content. The endophyte according to any aspect of the present invention acts as a biostimulant for the plant and / or part thereof with which it comes into contact. A more detailed definition of a biostimulant is provided at least in Ricci, M., General Principles to Justify Plant Biostimulant Claims, Frontiers in Plant Science (2019), 10.

[0025] As used herein, the term 'introduction' refers to contacting and / or treating a plant or part thereof with an endophyte, wherein the endophyte is delivered to the plant. In particular, the endophyte is introduced into the plant or part thereof to promote the growth of the endophyte therein. Any method for introducing the endophyte according to any aspect of the present invention into a plant or part thereof can be used. For example, the endophyte can be sprayed onto the plant or part thereof or inoculated into the plant or part thereof. In particular, the endophyte can be inoculated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before they begin to grow in the plant. In one example, the endophyte can be sprayed onto the leaves of the plant (i.e., foliar application). In this example, the spraying process is only carried out in the early developmental stage of the plant (about six leaves), or is carried out multiple times (about six leaves and several stages of new / adult plants). Technicians are able to identify the best introduction process for use on plants. In particular, plants or parts thereof can be infected with endophytes by methods known in the art. More particularly, plants or parts thereof may be infected with the endophyte using a method selected from the group consisting of inoculation, spraying, breeding, crossing, hybridisation, transduction, transfection, transformation and / or gene targeting, and combinations thereof.

[0026] In particular, the endophyte according to any aspect of the present invention is a Paenibacillus xylanicum strain having accession number DSM 34353. In particular, the sequence of the strain has been divided into loci and comprises the nucleotide sequences of SEQ ID NOs: 1-70 and variants thereof. In particular, the sequence of the 16S ribosomal RNA is SEQ ID NO: 78 and / or SEQ ID NO: 41.

[0027] As used herein, the term "variant" comprises an amino acid or nucleic acid sequence that is at least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to a reference amino acid or nucleic acid sequence, respectively, wherein preferably, amino acids other than those essential for the function (e.g., catalytic activity of a protein) or folding or structure of the molecule are deleted, substituted or replaced by insertion, or essential amino acids are replaced in a conservative manner such that the biological activity of the reference sequence or a molecule derived therefrom is substantially preserved. The prior art includes algorithms that can be used to align two given nucleic acid or amino acid sequences and calculate the degree of identity, see Arthur Lesk (2008), Thompson et al., 1994 and Katoh et al., 2005. The term "variant" is synonymous with the term "homolog" and is used interchangeably. Such variants may be prepared by introducing deletions, insertions or substitutions into an amino acid or nucleic acid sequence, as well as fusions comprising such macromolecules or variants thereof. In one example, in addition to the above-mentioned sequence identity, the term "variant" with respect to an amino acid sequence also encompasses an amino acid sequence comprising one or more conservative amino acid changes with respect to the respective reference or wild-type sequence, or comprises a nucleic acid sequence encoding an amino acid sequence comprising one or more conservative amino acid changes. In one example, in addition to the above-mentioned degree of sequence identity, the term "variant" with respect to an amino acid sequence or nucleic acid sequence also encompasses any active portion and / or fragment of an amino acid sequence or nucleic acid sequence, respectively, or any nucleic acid sequence encoding an active portion and / or fragment of an amino acid sequence. As used herein, the term "active portion" refers to an amino acid sequence or nucleic acid sequence that is less than the full-length amino acid sequence, respectively, or encodes less than the full-length amino acid sequence, wherein the amino acid sequence or the encoded amino acid sequence, respectively, retains at least some of its essential biological activity.

[0028] According to another aspect of the present invention, there is provided a biostimulant comprising an isolated endophyte, wherein the endophyte is a strain of Paenibacillus xylanicum having accession number DSM 34353. The endophyte is according to any aspect of the present invention.

[0029] According to a further aspect of the present invention, there is provided a composition comprising:

[0030] - an isolated endophyte, wherein the endophyte is a strain of Paenibacillus xylanicum having accession number DSM 34353, and

[0031] -Additives.

[0032] The compositions according to any aspect of the present invention address the problem of low uptake rates and kinetics of endophytes into the interior of plants. Adjuvants enable efficient uptake through stomata, small lesions in the cuticle, and growth cracks on the upper and, most importantly, lower surfaces of the plant. The use of adjuvants to support endophytic processes (penetration of microorganisms, particularly Paenibacillus xylans, into plant cells) allows for the use of lower drug concentrations (CFU / mL or CFU / g) because uptake occurs more efficiently than without the addition of adjuvants.

[0033] The endophyte was a Paenibacillus xylanicum strain with accession number DSM 34353.

[0034] The Paenibacillus xylans strains isolated according to any aspect of the present invention possess the appropriate genetic equipment to invade plant tissues and survive and proliferate within them. This, combined with suitable penetration sites on the surface of the plant and / or seed into which the Paenibacillus xylans strain is inoculated, enables the endophyte to successfully penetrate plant tissues via leaf stomata or via small wounds or growth cracks. Active penetration of non-uniformly shaped cuticles has also been reported.

[0035] However, as both the speed and kinetics of the endophytic process are limited in nature and therefore occur very slowly and host-specifically in most cases, the presence of an adjuvant in the composition according to any aspect of the invention allows for the endophyte according to any aspect of the invention to be successfully uptaken into (inside) plant tissue efficiently and effectively.

[0036] Typically, when Paenibacillus xylans is applied alone to soil, leaves, or seeds, the slow endophytic process results in the organism being exposed to biotic and abiotic stresses for a correspondingly long time. This can lead to a significant reduction in Paenibacillus xylans titer and, therefore, reduced product efficacy. The presence of an adjuvant according to any aspect of the present invention eliminates this negative effect and actually increases the efficacy of the composition by accelerating the uptake of the organism into the target plant or allowing it to be fully uptaken (to enable it at all).

[0037] As used herein, 'adjuvant' refers to an ingredient or substance in a composition according to any aspect of the present invention that increases or modifies the activity of another ingredient (i.e., the isolated Paenibacillus xylans strain). In particular, the adjuvant according to any aspect of the present invention is a biocompatible adjuvant (active ingredient medium) that is added to a suspension of microorganisms to form a composition according to any aspect of the present invention. Any adjuvant known in the art may be used in a composition according to any aspect of the present invention. In particular, the adjuvant according to any aspect of the present invention may be selected from (A), (B) or (C) and mixtures thereof, wherein (A), (B) or (C) is:

[0038] (A) a polyglycerol ester having the general formula (I),

[0039] M j D k T l Formula (I)

[0040] Wherein, M is [C3H5(OR”)2O 1 / 2 ],

[0041] D is [C3H5(OR”)1O 2 / 2 ],

[0042] T is [C3H5O 3 / 2 ],

[0043] j=1 to 10, preferably 2 to 3, more preferably 2;

[0044] k=0 to 10, preferably greater than 0 to 5, more preferably 1 to 3;

[0045] l=0 to 3, preferably 0 to 1, more preferably 0;

[0046] The sum of j+k+l is 1 to 20, preferably 2 to 4, and more preferably 3

[0047] wherein the radicals R" are each independently selected from the group consisting of acyl radicals R'-C(=O)- and H, with the proviso that at least one radical R" is not equal to H;

[0048] wherein the radicals R' are each independently selected from monovalent aliphatic, saturated or unsaturated hydrocarbon radicals having 3 to 39, preferably 7 to 21, more preferably 9 to 17 carbon atoms;

[0049] (B) Polyether-modified siloxane of formula (II)

[0050] M 1 o D 1 p D′ q Formula (II)

[0051] Among them, M 1 P 1 3SiO 1 / 2 ,

[0052] D 1 P 1 2SiO 2 / 2 , D′ is P 1 P 2 SiO 2 / 2 ,

[0053] o is 2, p is 0 to 0.1, q is 1.0 to 1.15,

[0054] P 1 are independently hydrocarbon groups having 1 to 8 carbon atoms,

[0055] P 2 are independently polyether groups of formula (III),

[0056] —P 3 O[CH2CH2O] m [CH2CH(CH3)O] n P 5 Formula (III)

[0057] in

[0058] m is 3.4 to 11.0, n is 2.5 to 8.0,

[0059] And the condition is

[0060] m / n is 1.9 to 2.8,

[0061] P 3 are independently divalent hydrocarbon groups having 2 to 8 carbon atoms,

[0062] P 5 is hydrogen; and / or

[0063] (C) Organic modified polysiloxane of formula (IV)

[0064] Formula (IV)

[0065]

[0066] Where a+b+c+d+2=20 to 210,

[0067] a is 15 to 205, b is 1 to 12, c is 1 to 12, d is 1 to 12,

[0068] The R groups are each independently the same or different aliphatic or aromatic hydrocarbon groups having 1 to 10 carbon atoms,

[0069] R4 groups are each independently the same or different R, R1, R2 or R3 groups,

[0070] R1, R2 and R3 groups are each independently different polyether groups of formula (V)

[0071] Formula (V)

[0072]

[0073] in

[0074] e is 3 to 11, f is 6 to 30, g is 0 to 15, h is 0 to 5, i is 0 to 5, and

[0075] R5 are independently the same or different and are each a methyl group, an acetyl group or a hydrogen group.

[0076] More particularly, the adjuvant may be selected from BREAK- S 301, BREAK- SP 133, BREAK- S255. The adjuvant used according to any aspect of the present invention leads to a reduction in surface tension at the stomata or at sites of damage therein, and thus to a lower rejection or improved flow of particles (microorganisms) through the pores into the plant or part thereof inoculated with the microorganisms, particularly Paenibacillus xylans. In particular, the use of the adjuvant allows / accelerates the endogenous process of bacterial uptake. In addition, the enhanced uptake also allows the translocation of bacteria from the leaves to the roots through the phloem, from where nitrogen fixation can also be enhanced. This results in the locally applied biostimulant acquiring systemic properties. Due to its systemic properties, there is the advantage that in plants with shoot formation, the shoots are also inoculated with the biostimulant, and thus the active ingredient is passed on to the new generation.

[0077] The use of adjuvants also allows the xylanicum bacteria to be evenly distributed from the upper surface of the leaves to the lower surface of the leaves where the majority of the (open) stomata are present. Thus, a faster and more extensive penetration of the endophyte into the plant tissue can be achieved. Without the use of a biocompatible adjuvant, especially in the case of foliar application, reaching the stomata on the underside of the leaves would be extremely difficult, if not impossible. The use of adjuvants with "anti-washout" properties also increases the residence time on the upper surface of the leaves and thus promotes the uptake of the endophyte into the plant tissue. The presence of the adjuvant reduces the early washout of endophytic nitrogen-fixing organisms.

[0078] In one embodiment, the adjuvant is (A):

[0079] (A) a polyglycerol ester having the general formula (I),

[0080] M j D k T l Formula (I)

[0081] Wherein, M is [C3H5(OR”)2O 1 / 2 ],

[0082] D is [C3H5(OR”)1O 2 / 2 ],

[0083] T is [C3H5O 3 / 2 ],

[0084] j=1 to 10, preferably 2 to 3, more preferably 2;

[0085] k=0 to 10, preferably greater than 0 to 5, more preferably 1 to 3;

[0086] l=0 to 3, preferably 0 to 1, more preferably 0;

[0087] The sum of j+k+l is 1 to 20, preferably 2 to 4, and more preferably 3

[0088] wherein the radicals R" are each independently selected from the group consisting of acyl radicals R'-C(=O)- and H, with the proviso that at least one radical R" is not equal to H;

[0089] The radicals R' are each independently selected from monovalent aliphatic, saturated or unsaturated hydrocarbon radicals having 3 to 39, preferably 7 to 21, more preferably 9 to 17 carbon atoms.

[0090] In particular, at least one radical R" corresponds to a radical of the formula R'-C(O)-.

[0091] More specifically, M, D, and T may be:

[0092]

[0093] Even more particularly, the polyglycerol ester of the mixture according to any aspect of the invention has formula (I(a)):

[0094]

[0095] in

[0096] a=1 to 10, preferably 2 to 3, more particularly 2;

[0097] b=0 to 10, preferably greater than 0 to 5, more particularly 1 to 3;

[0098] The conditions are:

[0099] a+b=2 to 20, preferably 2 to 4, especially 3;

[0100] wherein the radicals R" are each independently selected from acyl radicals R'-C(=O)- and H, with the proviso that at least one radical R is not equal to H; wherein the radicals R' are each independently selected from monovalent aliphatic, saturated or unsaturated hydrocarbon radicals having 3 to 39, preferably 7 to 21, in particular 9 to 17 carbon atoms.

[0101] The polyglycerol ester of the composition according to any aspect of the invention may have more than one, in particular at least 2, more in particular at least 3 groups R" of the form R'-C(=O)-.

[0102] The radicals R" of the formula R'-C(O)- can independently be identical or different acyl radicals of saturated or unsaturated fatty acids, wherein the fatty acids comprise 4 to 40 carbon atoms, in particular fatty acids selected from butyric acid (butanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid), acid), lauric acid (dodecanoic acid), myristic acid (tetradecanoic acid), palmitic acid (hexadecanoic acid), stearic acid (octadecanoic acid), arachidic acid (eicosanoic acid), behenic acid (docosanoic acid), lignoceric acid (tetradecanoic acid), palmitoleic acid ((Z)-9-hexadecenoic acid), oleic acid ((Z)-9-hexadecenoic acid), elaidic acid ((E)-9-octadecenoic acid), cis-vaccenic acid ((Z)-11-octadecenoic acid), linoleic acid ((9Z,12Z)-9,12-octadecadienoic acid), α-linolenic acid ((9Z,12Z,15Z)-9,12,15-octadecatrienoic acid), γ-linolenic acid ((6Z,9Z,12Z)-6,9,12-octadecatrienoic acid), dihomo-γ- Linolenic acid ((8Z,11Z,14Z)-8,11,14-eicosatrienoic acid), arachidonic acid ((5Z,8Z,11Z,14Z)-5,8,11,14-eicosatrienoic acid), erucic acid ((Z)-13-docosaenoic acid), nervonic acid ((Z)-15-tetracosenoic acid), ricinoleic acid, hydroxystearic acid, undecenoic acid, and mixtures thereof. In one example, the fatty acid may be a mixture of rapeseed oil acid, soybean fatty acid, sunflower fatty acid, peanut fatty acid, and tall oil fatty acid. In particular, for this context, the fatty acid may be a radical of oleic acid. When calculating the HLB value, the molar mass of the lipophilic portion of the molecule is the arithmetic mean of the sum of the molar masses of all groups R' present in the molecule.

[0103] Suitable sources of fatty acids or fatty acid esters, in particular glycerides, may be vegetable or animal fats, oils or waxes. For example, lard, beef tallow, goose fat, duck fat, chicken fat, horse fat, whale oil, fish oil, palm oil, olive oil, avocado oil, seed oil, coconut oil, palm kernel oil, cocoa butter, cottonseed oil, pumpkin seed oil, corn seed oil, sunflower oil, wheat germ oil, grape seed oil, soybean oil, peanut oil, lupine oil, rapeseed oil, mustard oil, castor oil, jatropa oil, Oil), walnut oil, jojoba oil, lecithin (e.g. based on soybean, rapeseed or sunflower), bone oil, claw oil, borage oil, lanolin, emu oil, deer tallow, woodchuck oil, mink oil, safflower oil, hemp oil, pumpkin oil, evening primrose oil, tall oil, as well as carnauba wax, beeswax, candelilla wax, ouricuri wax, sugarcane wax, retamow wax, caranday wax, raffia wax, esparto grass wax, alfalfa wax, bamboo wax, hemp wax, Douglas fir wax, cork wax, sisal wax, flax wax, cotton wax, dammar wax, tea wax, coffee wax, rice wax, oleander wax or wool wax can be a source of fatty acids or fatty acid esters.

[0104] In particular, the polyglycerol ester compound has the formula (I), or (I(a)) having an arithmetic mean of 2.9 to 3.1 groups of the form R'-C(=0)- and an HLB value of 4 to 6.5.

[0105] More particularly, the polyglycerol ester compound has the formula (I(a)) having a total a+b of 3, an arithmetic mean of 2.9 to 3.1 groups of the form R'-C(=0)- and an HLB value of 4 to 6.5.

[0106] Even more particularly, the polyglycerol ester compound may have formula (I(a)) having an arithmetic mean of 2.9 to 3.1 groups of the form R'-C(=0)- and an HLB value of 4 to 6.5, wherein the acyl residues are of a fatty acid mixture containing oleic acid, stearic acid, palmitic acid and gamma-linolenic acid, and said fatty acids in particular represent at least 85% by weight of the fatty acid mixture.

[0107] In one example, the polyglycerol ester compound can have the formula (I(a)) having an arithmetic mean of 2.9 to 3.1 groups of the form R'-C(=0)- and an HLB value of 4 to 6.5, the acyl residues being derived from a fatty acid mixture containing oleic acid, stearic acid, palmitic acid and γ-linolenic acid, and the fatty acids in particular representing at least 85% by weight of the fatty acid mixture.

[0108] In another example, the polyglycerol ester compound used according to any aspect of the present invention may have the formula (I(a)), which has an arithmetic mean of 2.9 to 3.1 groups of the form R'-C(=O)- and an HLB value of 4 to 6.5, and the mass fraction of oleic acid acyl residues is at least 75%, particularly 85%, more particularly 95% based on the mass of all acyl residues. Even more particularly, the polyglycerol ester is triglycerol trioleate.

[0109] A more comprehensive disclosure of adjuvants (B) is provided at least in US Pat. No. 1,039,530 B2.

[0110] In one embodiment, the adjuvant is (B):

[0111] (B) Polyether-modified siloxane of formula (II)

[0112] M 1 o D 1 p D′ q Formula (II)

[0113] Among them, M 1 P 1 3SiO 1 / 2 ,

[0114] D 1 P 1 2SiO 2 / 2 , D′ is P 1 P 2 SiO 2 / 2 ,

[0115] o is 2,

[0116] p is 0 to 0.1, in particular 0

[0117] q is from 1.0 to 1.15, in particular from 1.0 to 1.10, and very particularly from 1.00 to 1.05,

[0118] P 1 is independently a hydrocarbon radical having 1 to 8 carbon atoms, in particular a methyl, ethyl, propyl or phenyl radical, particularly in particular a methyl radical,

[0119] P 2 are independently polyether groups of formula (III),

[0120] —P 3 O[CH2CH2O] m [CH2CH(CH3)O] n P 5 Formula (III)

[0121] in

[0122] m is from 3.4 to 11.0, in particular from 3.6 to 9.9, more in particular from 4.5 to 8.5,

[0123] n is 2.5 to 8.0, particularly 2.7 to 7.5, more particularly 3.0 to 6.0,

[0124] And the condition is

[0125] m / n is 1.9 to 2.8,

[0126] P 3 is independently a divalent hydrocarbon radical having 2 to 8 carbon atoms, in particular an ethylene, propylene, 1-methylpropylene, 1,1-dimethylpropylene radical, and especially in particular —CH2CH2CH2—,

[0127] P 5 For hydrogen.

[0128] The polyether-modified siloxanes of formula (II) have a biodegradability of greater than 60%, more particularly greater than or equal to 63% and very particularly greater than or equal to 65%, with a maximum of 100%.

[0129] In particular, no P 3 O calculation and does not include P 5 The calculated polyether group has a molar mass M(PE) calculated as 44 g / mol*m+58 g / mol*n, where the indices m and n are related to formula (III). More particularly, the value of M(PE) is: the lower limit M(PE) is greater than 520 g / mol, particularly greater than 530 g / mol, more particularly greater than 535 g / mol; the upper limit M(PE) is less than 660 g / mol, particularly less than 630 g / mol, more particularly less than 600 g / mol. Even more particularly, the value of M(PE) is greater than 520 g / mol and less than 660 g / mol, especially greater than 535 g / mol and less than 600 g / mol. In particular, the sum of m+n is greater than 9 up to 19, more particularly greater than 9.5 up to 15, and even more particularly greater than 10 up to 12.

[0130] In one example, the polyether-modified siloxane used in the composition according to any aspect of the present invention is a polyether-modified siloxane of formula (II) wherein the index c is from 1 to 1.05, wherein the index of the polyether group of formula (III) is m from 3.4 to 11.0 and n from 2.5 to 8.0. In particular, the polyether-modified siloxane used in the composition according to any aspect of the present invention is a polyether-modified siloxane of formula (II) wherein the index c is from 1 to 1.05, wherein the ratio m / n is from 1.9 to 2.8. More particularly, the polyether-modified siloxane used in the composition according to any aspect of the present invention is a polyether-modified siloxane of formula (II) wherein the index c is from 1 to 1.05, wherein the molar mass of the polyether residue M(PE) is greater than 520 g / mol and less than 660 g / mol. Even more particularly, the polyether-modified siloxane used in the composition according to any aspect of the present invention is a polyether-modified siloxane of formula (II) wherein the index c is from 1 to 1.05, wherein P 5 The group is hydrogen, or wherein the index c is between 1 and 1.05, wherein the molar mass of the polyether residue M(PE) is greater than 520 g / mol and less than 660 g / mol and P 5 The group is hydrogen. In particular, the polyether-modified silicone used in the composition according to any aspect of the present invention is a polyether-modified silicone of formula (II) and does not include any further polyether-modified silicones other than those of formula (II).

[0131] A more comprehensive disclosure of adjuvants (B) is provided at least in US 10299471 B2.

[0132] In one embodiment, the auxiliary agent is (C), an organic modified polysiloxane of formula (IV)

[0133] Formula (IV)

[0134]

[0135] wherein a+b+c+d+2=20 to 210, preferably 30 to 100, especially 40-60,

[0136] a is 15 to 205, preferably 35 to 45,

[0137] b is 1 to 12, preferably 1 to 8, especially 2 to 6,

[0138] c is 1 to 12, preferably 1 to 8, especially 2 to 6,

[0139] d is 1 to 12, preferably 1 to 8, especially 2 to 6,

[0140] The R groups are each independently the same or different aliphatic or aromatic hydrocarbon groups having 1 to 10 carbon atoms, preferably a methyl group,

[0141] R4 groups are each independently the same or different R, R1, R2 or R3 groups,

[0142] R1, R2 and R3 groups are each independently different polyether groups of formula (V)

[0143] Formula (V)

[0144]

[0145] in

[0146] e is 3 to 11, preferably 3,

[0147] f is 6 to 30, preferably 10 to 30,

[0148] g is 0 to 15, preferably 0 to 10,

[0149] h is 0 to 5,

[0150] i is 0 to 5, and

[0151] R5 are independently identical or different and are each a methyl, acetyl or hydrogen radical, preferably with the proviso that the molecular weight of the polyether radical of formula (V) is greater than 200 g / mol, preferably greater than 400 to 2000 g / mol, and the proportion of ethylene oxide is greater than 45% by mass of the polyether, and the percentage by mass of ethylene oxide in the polyether radical R2 is at least 9% greater by mass than the percentage by mass of ethylene oxide in the polyether radical R1, in each case based on the polyether radical of formula (V), wherein the radicals of formula (V) can each be formed randomly, in a gradient or in blocks.

[0152] In formula (V), the units designated by the index 'g' are those derived from propylene oxide, the units designated by the index 'h' are those derived from butylene oxide, and the units designated by the index 'i' are those derived from styrene oxide.

[0153] The indices 'a to d' and 'e to i' may be natural integers or weighted averages. The indices are preferably weighted averages.

[0154] A more comprehensive disclosure of auxiliaries (C) is provided at least in US Pat. No. 8,580,225 B2.

[0155] The composition according to any aspect of the present invention may comprise any one of the adjuvants (A), (B), or (C). In one example, the composition may comprise a mixture of adjuvants, such as (A) and (B), (A) and (C), (B) and (C), or (A), (B), and (C). In another example, the composition according to any aspect of the present invention may comprise more than one adjuvant (A), more than one adjuvant (B), or more than one adjuvant (C).

[0156] The composition according to any aspect of the present invention may further comprise an emulsifier. The emulsifier in the mixture according to any aspect of the present invention may be different from the adjuvant. The emulsifier may be selected from fatty acid esters of polyols and polyalkylene glycol derivatives thereof, polyethylene glycol derivatives of fatty acids and fatty alcohols, sorbitan fatty acid esters, ethoxylated and / or propoxylated sorbitan fatty acid esters, propoxylated sorbitan fatty acid esters, alkylphenol polyoxyethylene ethers, propoxylates, alkylphenol polyoxyethylene ethers, aminoxylated oxides, amine oxides, propoxylated amine oxides, aminooxylated amine oxides, aminooxylated propylene oxides, acetylene glycol surfactants, ethoxylated and / or propoxylated acetylene glycols, silicone surfactants and mixtures thereof. In particular, the emulsifier is selected from sorbitan fatty acid esters and ethoxylated sorbitan fatty acid esters. More particularly, the emulsifier is ethoxylated sorbitan fatty acid esters or mixtures thereof.

[0157] The acyloxy groups of the sorbitan fatty acid esters or ethoxylated sorbitan fatty acid esters have 4 to 40, in particular 8 to 22, more particularly 10 to 18, carbon atoms, and / or the sorbitan fatty acid esters or ethoxylated sorbitan fatty acid esters have 0 to 40, in particular 10 to 30, more particularly 15 to 25 oxyethylene groups. The fatty acids or fatty acid residues of the sorbitan fatty acid esters are particularly defined as the fatty acids or fatty acid residues of the polyglycerol esters. The acyl groups (also called alkanoyl groups) are particularly derived from a fatty acid mixture containing oleic acid, stearic acid, palmitic acid, and gamma-linolenic acid, the fatty acids particularly comprising at least 85% by weight of the fatty acid mixture. In particular, ethoxylated sorbitan fatty acid esters are used in which the mass fraction of oleic acid acyl residues, based on the mass of all acyl residues, is at least 75%, in particular 85%, more particularly 95%.

[0158] The emulsifier according to any aspect of the invention has an HLB value greater than or equal to 9, in particular greater than or equal to 10, more particularly greater than or equal to 11. The HLB value may be a maximum of 16, more particularly a maximum of 15, even more particularly a maximum of 13. In particular, the emulsifier has an HLB value of 9 to 16, in particular from 10 to 15, more particularly from 11 to 13. The HLB value is determined as described above. The HLB value of the sorbitan fatty acid ester and / or ethoxylated sorbitan fatty acid ester is in particular determined as for the polyglycerol ester. The molar mass of the lipophilic part of the molecule results from the arithmetic mean of the sum of the molar masses of all groups R'' present as part of the acyl group R''-(CO)- in the molecule. The radical R'' is preferably as defined for the polyglycerol esters. The radical R'' as part of the acyl group R''-(CO)- of the sorbitan fatty acid ester or ethoxylated sorbitan fatty acid ester is in particular chosen from monovalent aliphatic, saturated or unsaturated hydrocarbon radicals having 3 to 39, preferably 7 to 21, in particular 9 to 17 carbon atoms. The calculation of the molar mass of the entire molecule is carried out as defined above. In particular, the emulsifier is polyethylene glycol-20-sorbitan trioleate. The number 20 indicates the average number of ethylene oxide units in the polyethylene glycol residue.

[0159] The HLB values of the polyglycerol ester and the emulsifier are matched to each other. The polyglycerol ester has an HLB value of less than or equal to 8, particularly less than or equal to 7, more particularly less than or equal to 6.5, and the at least one emulsifier has an HLB value greater than or equal to 9, particularly greater than or equal to 10, particularly greater than or equal to 11. The at least one polyglycerol ester has an HLB value of 0.5 to 8, particularly 1 to 7, more particularly 2 to 6.5, and the at least one emulsifier has an HLB value of 9 to 16, particularly 10 to 15, more particularly 11 to 13. In particular, the emulsifier is polyethylene glycol 20 sorbitan trioleate.

[0160] According to a further aspect of the present invention, there is provided a culture medium comprising an isolated endophyte and an endophyte composition, wherein the endophyte is a strain of Paenibacillus xylanicum having accession number DSM 34353.

[0161] The strains and compositions according to any aspect of the present invention can be obtained by culturing a strain of Paenibacillus xylans using an appropriate culture medium according to methods well known in the art. Conventional large-scale microbial culture methods include submerged fermentation, solid-state fermentation, or liquid surface culture. Endophytes can be cultured under aerobic or anaerobic conditions and can be cultured in bioreactors. Endophytes and metabolites in the culture medium resulting from the culture can be used directly or concentrated by conventional industrial methods such as centrifugation, tangential flow filtration, deep filtration, and evaporation. The concentrated fermentation broth can be washed, for example, via a diafiltration process to remove residual fermentation broth and metabolites.

[0162] The fermented liquid or fermented liquid concentrate may be dried with or without the addition of a carrier, using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized bed drying, drum drying or evaporation. The resulting dried product can be further processed, for example, by grinding or granulation, to achieve a specific particle size or physical form. A carrier can also be added after drying. In particular, the preparation of the bacterial strain is the supernatant of the fermented liquid. In an example, the composition according to any aspect of the present invention can be prepared according to the method provided in EP21198571 or EP21202623, wherein the endophyte according to any aspect of the present invention is first spray dried and then contacted with at least one auxiliary agent. In particular, the auxiliary agent can be (A), (B) or (C) according to any aspect of the present invention. More particularly, the auxiliary agent is (A), a polyglycerol ester with general formula (I). Even more particularly, the polyglycerol ester is combined with at least one emulsifier according to any aspect of the present invention.

[0163] According to a further aspect of the present invention, there is provided a plant or part thereof infected with one or more endophytes according to any aspect of the present invention. In particular, the plant or part thereof infected with the endophyte may produce a bioprotectant compound. In particular, the plant or part thereof includes an endophyte-free host plant or part thereof stably infected with the endophyte.

[0164] Any plant or part thereof can be inoculated with the endophyte according to any aspect of the present invention. In particular, the plant inoculated with the endophyte can be an herbaceous plant or non-herbaceous plant suitable for agriculture, in particular forage grass, turf grass or bioenergy grass, or a cereal crop or industrial crop. More particularly, the cereal crop or industrial crop species can be selected from wheat, barley, oats, corn / maize, any cereal legume such as chickpea, triticale, broad bean, lupine, field pea, rape (canola), cereal rye, vetch, lentil, chestnut / millet, safflower, linseed, sorghum, sunflower, maize, rape, mung bean, soybean, oilseed crops, tomato and cotton.

[0165] Endophytes according to any aspect of the present invention can be transferred from one plant generation to the next via seeds. The endophytes can then spread or localize to other tissues, such as roots, as the plant grows. Alternatively or additionally, the endophytes can be recruited to plant roots, for example, from the soil, and spread or localize to other tissues. According to a further aspect of the present invention, plants, plant seeds, or other plant parts derived from plants according to any aspect of the present invention, or parts thereof, are provided. The plants, plant seeds, or other plant parts can produce bioprotectant compounds.

[0166] Endophyte-infected plants or parts thereof can be cultivated by known techniques. A person skilled in the art can easily determine appropriate conditions depending on the plant or part thereof to be cultivated.

[0167] According to another aspect of the present invention, there is provided a method of producing a composition for bioprotection and / or biostimulation, the method comprising combining:

[0168] - an isolated endophyte, wherein the endophyte is a strain of Paenibacillus xylanicum, and

[0169] -Additives.

[0170] According to a further aspect of the present invention, there is provided a method of providing biological protection to a plant or a part thereof, the method comprising contacting the plant or a part thereof with a composition according to any aspect of the present invention.

[0171] The part of a plant may be, for example, a seed. In one example, the composition according to any aspect of the present invention is introduced into the leaves, roots or seeds of a plant. In particular, the composition according to any aspect of the present invention is introduced into the leaves of a plant. Applying endophytes to leaves allows the user (if necessary) to process growing plants or adult plants at several different times after sowing. By contrast, during soil application (before sowing) or seed application (during seed treatment), only a single application is possible. Further, foliar application of Paenibacillus xylans allows the use of microbial agents to be reduced because, when inoculating seeds or soil, higher CFU losses (several log levels) can be expected in a short period of time (the stability of Gram-negative organisms on seeds or in soil is lower).

[0172] According to a further aspect of the present invention there is provided use of a composition according to any aspect of the present invention for providing bioprotection to a plant or part thereof. Example

[0173] The foregoing describes preferred embodiments, which, as will be appreciated by those skilled in the art, may be subject to changes or modifications in design, construction, or operation without departing from the scope of the claims. For example, these changes are intended to be encompassed by the scope of the claims.

[0174] Example 1

[0175] Strain collection

[0176] For the collection of the Paenibacillus xylans strain with accession number DSM 34353, different plants were collected from different locations. The locations were selected based on their unique habitats. A list of the locations and plant types used to collect Paenibacillus xylans DSM 34353 is provided in Table 1.

[0177]

[0178] Table 1. List of locations and plants used to collect Paenibacillus xylans DSM 34353

[0179] A trowel or small spade was used to gently dig up the soil around each plant and lift the roots with minimal disturbance. Root sections were removed gently, without damaging the entire root system. Similarly, leaf material was harvested from each plant without damaging the plant. Plant material was placed in separate plastic bags, which were then carefully packed into insulated, refrigerated transport bags. After collection on site, plant material was refrigerated at 5°C until further use.

[0180] As part of pretreatment, leaves and roots of each plant were washed in sterile distilled water under slow running tap water for 15 minutes to remove adhering soil particles and most microbial epiphytes. The samples were then rinsed three times with sterile distilled water in a laminar flow cabinet for one minute each.

[0181] Cut the roots and leaves into segments. Place the plant material in a Petri dish, soak it in distilled water, and drain. Rinse it in 70% ethanol for 30 seconds, then sterilize it with 3% sodium hypochlorite for 3 minutes for roots and 5 minutes for leaves. The tissue is then washed ten times with sterile water. Validate the surface sterilization procedure by culturing an aliquot of the water from the final rinse on nutrient medium.

[0182] After the surface-sterilized plant material was properly dried in a laminar flow cabinet, the roots and leaves were cut into small pieces 2-3 cm long. The outer part of the leaf, approximately 0.5 cm from the edge, was removed using a sterile razor blade. Each root and leaf piece was placed on nutrient agar medium supplemented with 100 mg L-1 of cycloheximide to inhibit possible fungal development. The plates containing the plant tissue were sealed with parafilm tape and incubated at 28±2°C to recover as many bacterial endophyte colonies as possible. After 48 hours, morphologically distinct bacterial colonies were selected from the root tips and leaf nodes and repeatedly streaked after four-fold serial dilution to obtain enriched bacterial isolates. To obtain pure cultures, the enriched cultures were streaked onto individual 50% TS agar plates and incubated at 30°C for 2-3 days.

[0183] The purity of the culture was checked by visual examination of colony morphology. Colonies that appeared different were separated and streaked again on separate 50% TS agar plates for new cultivation. In this way, the mixed culture was divided into monoseptic isolates. Purity was confirmed by microscopic analysis and subsequent comparison of the 16S sequence with the corresponding sequence from the NCBI rRNA / ITS database using "blastn". The latter program is provided as a service by VERMICON (Hallbergmoos, Germany).

[0184] Cell material from these re-plated cultures was resuspended in 50% glycerol solution, as was done with the primary cell material, and stored at -80°C. To test re-vitalizing, new cultures were inoculated from these frozen stocks. Growing cultures were labeled "pure" and "cryopreserved." For genome sequencing, strains of particular interest were grown in liquid culture, centrifuged, and resuspended in 70% ethanol. Approximately 1 x 10 8 Cells from these strains were sent to LGC Biomics (Berlin, Germany) for whole-genome sequencing. It was confirmed to be a novel strain. The strain is Paenibacillus xylanicum DSM 34353.

[0185] Example 2

[0186] Efficacy analysis

[0187] To determine the nitrogen-fixing efficacy of Paenibacillus xylanicum DSM 34353 as an endogenous biostimulant, untreated maize seeds (Zea mays, variety LG 31.224) were planted in a poor, sandy soil (Oxisol) with little organic matter (importantly low in nitrogen, so that the effects of free-living nitrogen-fixing bacteria become apparent).

[0188] Five seeds were planted per pot, which were further thinned to three plants after germination to avoid a low number of replicates. Fertilizer dosage rates were calculated based on 1,000,000 kg soil / ha. Fertilization was carried out with phosphate, potassium, and magnesium sulfate (25 kg P / ha, 50 kg K / ha, and 15 kg Mg / ha, respectively). A water-soluble 21% N and 24% S (24 and 48 kg N / ha) Fertilization with nitrogen was performed using 45 (Domo, Germany). 2 L pots were used and filled with 2 kg of sandy soil.

[0189] At the sixth leaf stage (approximately 2 weeks after planting), Paenibacillus xylanexedens DSM 34353 was applied to the leaves in combination with an adjuvant. During the process, a spray volume of 1 mL was applied to the leaves of young maize plants. The mixture contained 300.000 CFU of Paenibacillus xylanexedens DSM 34353 and 0.1% of (Evonik Industries, Germany) for surface wetting and pore flooding. The auxiliary agent (B) according to any aspect of the present invention has the formula. In particular, With the following formula

[0190]

[0191] A 1 mL spray of the leaves of the pots corresponds to approximately 400 L spray / ha, which is similar to the standard application rate for field application. The maize plants were grown at a temperature of 20 to 25°C with regular irrigation. Pictures ( Figure 1 and 2 ), and the root ( Figure 4 ) and branches ( Figure 3 ) of dry mass.

[0192] Shown with additives The efficacy of the combined Paenibacillus xylans DSM 34353 was excellent.

[0193] Furthermore, mixtures with other microorganisms such as the biofertilizer Blue-N (Corteva, Inc, USA) with Methylobacterium symbioticum, Kreotec (Biofa) with Bacillus velezensis, Azospirillum brasilense and Herbaspirillum seropedicae, the biofertilizer Utrisha with Methylobacterium symbioticum SB23 TM -N (Corteva, Inc, USA), Blue-N (Corteva, Inc, USA) with Methylobacterium symbioticum, Blue-N (Corteva, Inc, USA) with Bacillus atrophaeus (AbiTep, Germany) and RhizoFos (Rizobacter, USA) with Pseudomonas fluorescens were used as controls for comparative studies. These comparator products also contained microorganisms capable of fixing atmospheric nitrogen and were also applied to the leaves. However, using reduced anthropogenic nitrogen fertilization (21 kg N / ha), the comparator products showed no or a lower growth-promoting effect.

[0194] These comparative studies have shown that the use of Utrisha TM The use of Paenibacillus xylans in combination with adjuvants resulted in better plant growth under nitrogen-limited conditions compared to using either Aqua-N, Blue-N or Kreotec.

[0195] Example 3

[0196] Effects of different mineral nitrogen fertilizer dosages on the efficacy of nitrogen-fixing bacterial endophyte Paenibacillus xylanicum

[0197] The overall aim of Example 3 was to find out how much nitrogen the bacterial endophyte Paenibacillus xylanicum could fix under field conditions, ie under different mineral fertilizer dosages for crop selectivity and yield (grain and shoots) under field conditions.

[0198] When treating seeds, the bacterial endophyte was applied via wet seed treatment. When applying foliarly, the bacterial endophyte was applied at the 5-6 leaf stage.

[0199] Corn seed treatment

[0200] Planting 100,000 seeds, apply 1,000,000 CFU (bacterial units) per seed. At 100,000 seeds / ha: 1x10 11 CFU / ha

[0201] Each bacterial suspension had 2.5 x 10 8 CFU / mL, so 400 mL of experimental product is required for every 100 000 pellets. This results in 4 mL of experimental product per 1000 pellets (with 2.5 x 10 8 CFU). A mixture of 4 mL of the test product, 1 mL of PREMAX, and pickle was prepared for 1000 corn kernels, and then left to dry in the dark (e.g., by opening a paper bag). For the control, nothing was added.

[0202] Foliar treatment

[0203] 50,000 CFU (units) of the experimental bacteria were added per plant. The plants were at the 5-6 leaf stage, where only about 10% or less of the soil area was covered by plants. Using 100,000 plants / ha and 50,000 CFU, it resulted in a total of 5x10 9 CFU / 10% area of 1ha - that is, 5 x 10 10 CFU / ha. Spray 200 mL / ha with 2.5 x 10 8 CFU / mL of experimental bacterial product - this results in 5x10 10 CFU / ha.

[0204] Dilute 10 mL of the test product (2.5 x 10 8 CFU / mL) - this gives 2.5x10 7 CFU / mL. In this 100mL (with 2.5x 10 7 CFU / mL), apply 20mL / 100m 2 - or 2000 mL / ha. The water application rate (spray liquid) was 300 L / ha and BREAK- SP 133: 300 mL / ha (= 0.1%). All experiments were conducted in Germany.

[0205] The following experiments were conducted:

[0206]

[0207] The results are provided in Table 2 below and Figure 5.

[0208]

[0209] Table 2. Results of Example 3

[0210] The results showed that the overall yield increased when the biostimulant (ie Paenibacillus xylanicum) was applied in addition to the synthetic fertilizer.

[0211]

Claims

1. A composition comprising: - an isolated endophyte, wherein the endophyte is a strain of Paenibacillus xylanexedens, and - additives, The Paenibacillus xylans strain has the accession number DSM 34353.

2. The composition according to claim 1, wherein the adjuvant is selected from (A), (B) and (C): (A) a polyglycerol ester having the general formula (I), M j D k T l Formula (I) in, M is [C3H5(OR”)2O 1 / 2 ], D is [C3H5(OR”)1O 2 / 2 ], T is [C3H5O 3 / 2 ], j=1 to 10, preferably 2 to 3, more preferably 2; k=0 to 10, preferably greater than 0 to 5, more preferably 1 to 3; l=0 to 3, preferably 0 to 1, more preferably 0; The sum of j+k+l is 1 to 20, preferably 2 to 4, and more preferably 3 wherein the radicals R" are each independently selected from the group consisting of acyl radicals R'-C(=O)- and H, with the proviso that at least one radical R" is not equal to H; wherein the radicals R' are each independently selected from monovalent aliphatic, saturated or unsaturated hydrocarbon radicals having 3 to 39, preferably 7 to 21, more preferably 9 to 17 carbon atoms; (B) Polyether-modified siloxane of formula (II) M 1 o D 1 p D′ q Formula (II) Among them, M 1 P 1 3SiO 1 / 2 , D 1 P 1 2SiO 2 / 2 , D′ is P 1 P 2 SiO 2 / 2 , o is 2, p is 0 to 0.1, q is 1.0 to 1.15, P 1 are independently hydrocarbon groups having 1 to 8 carbon atoms, P 2 are independently polyether groups of formula (III), —P 3 O[CH2CH2O] m [CH2CH(CH3)O] n P 5 Formula (III) in m is 3.4 to 11.0, n is 2.5 to 8.0, And the condition is m / n is 1.9 to 2.8, P 3 are independently divalent hydrocarbon groups having 2 to 8 carbon atoms, P 5 is hydrogen; and / or (C) Organic modified polysiloxane of formula (IV) Formula (IV) Where a+b+c+d+2=20 to 210, a is 15 to 205, b is 1 to 12, c is 1 to 12, d is 1 to 12, The R groups are each independently the same or different aliphatic or aromatic hydrocarbon groups having 1 to 10 carbon atoms, R4 groups are each independently the same or different R, R1, R2 or R3 groups, R1, R2 and R3 groups are each independently different polyether groups of formula (V) Formula (V) in e is 3 to 11, f is 6 to 30, g is 0 to 15, h is 0 to 5, i is 0 to 5, and R5 are independently the same or different and are each a methyl group, an acetyl group or a hydrogen group.

3. The composition according to claim 1 or 2, further comprising at least one emulsifier.

4. The composition of claim 3, wherein the emulsifier is selected from the group consisting of sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, and mixtures thereof.

5. A substantially purified or isolated endophyte, wherein the endophyte is a strain of Paenibacillus xylanicum that provides a bioprotective and / or biostimulant phenotype to a plant into which the endophyte is introduced, and wherein the endophyte Paenibacillus xylanicum strain has accession number DSM 34353.

6. The endophyte of claim 5, wherein the bioprotection and / or biostimulant phenotype comprises nitrogen fixation in a plant into which the endophyte is introduced.

7. A biostimulant comprising an isolated endophyte, wherein the endophyte is a strain of Paenibacillus xylanicum having accession number DSM 34353.

8. The biostimulant according to claim 7, further comprising at least one adjuvant selected from (A), (B) and (C): (A) a polyglycerol ester having the general formula (I), M j D k T l Formula (I) in, M is [C3H5(OR”)2O 1 / 2 ], D is [C3H5(OR”)1O 2 / 2 ], T is [C3H5O 3 / 2 ], j=1 to 10, preferably 2 to 3, more preferably 2; k=0 to 10, preferably greater than 0 to 5, more preferably 1 to 3; l=0 to 3, preferably 0 to 1, more preferably 0; The sum of j+k+l is 1 to 20, preferably 2 to 4, and more preferably 3 wherein the radicals R" are each independently selected from the group consisting of acyl radicals R'-C(=O)- and H, with the proviso that at least one radical R" is not equal to H; wherein the radicals R' are each independently selected from monovalent aliphatic, saturated or unsaturated hydrocarbon radicals having 3 to 39, preferably 7 to 21, more preferably 9 to 17 carbon atoms; (B) Polyether-modified siloxane of formula (II) M 1 o D 1 p D′ q Formula (II) Among them, M 1 P 1 3SiO 1 / 2 , D 1 P 1 2SiO 2 / 2 , D′ is P 1 P 2 SiO 2 / 2 , o is 2, p is 0 to 0.1, q is 1.0 to 1.15, P 1 are independently hydrocarbon groups having 1 to 8 carbon atoms, P 2 are independently polyether groups of formula (III), —P 3 O[CH2CH2O] m [CH2CH(CH3)O] n P 5 Formula (III) in m is 3.4 to 11.0, n is 2.5 to 8.0, And the condition is m / n is 1.9 to 2.8, P 3 are independently divalent hydrocarbon groups having 2 to 8 carbon atoms, P 5 is hydrogen; and / or (C) Organic modified polysiloxane of formula (IV) Formula (IV) Where a+b+c+d+2=20 to 210, a is 15 to 205, b is 1 to 12, c is 1 to 12, d is 1 to 12, The R groups are each independently the same or different aliphatic or aromatic hydrocarbon groups having 1 to 10 carbon atoms, R4 groups are each independently the same or different R, R1, R2 or R3 groups, R1, R2 and R3 groups are each independently different polyether groups of formula (V) Formula (V) in e is 3 to 11, f is 6 to 30, g is 0 to 15, h is 0 to 5, i is 0 to 5, and R5 are independently the same or different and are each a methyl group, an acetyl group or a hydrogen group.

9. A composition comprising a culture medium of an isolated endophyte, wherein the endophyte is a strain of Paenibacillus xylanicum having accession number DSM 34353.

10. A method of producing a composition for bioprotection and / or biostimulation, the method comprising combining: - an isolated endophyte, wherein the endophyte is a strain of Paenibacillus xylanicum, and - additives, The strain of Paenibacillus xylans has the accession number DSM 34353.

11. The method according to claim 10, wherein the auxiliary agent is selected from (A), (B) or (C): (A) a polyglycerol ester having the general formula (I), M j D k T l Formula (I) in, M is [C3H5(OR”)2O 1 / 2 ], D is [C3H5(OR”)1O 2 / 2 ], T is [C3H5O 3 / 2 ], j=1 to 10, preferably 2 to 3, more preferably 2; k=0 to 10, preferably greater than 0 to 5, more preferably 1 to 3; l=0 to 3, preferably 0 to 1, more preferably 0; The sum of j+k+l is 1 to 20, preferably 2 to 4, and more preferably 3 wherein the radicals R" are each independently selected from the group consisting of acyl radicals R'-C(=O)- and H, with the proviso that at least one radical R" is not equal to H; wherein the radicals R' are each independently selected from monovalent aliphatic, saturated or unsaturated hydrocarbon radicals having 3 to 39, preferably 7 to 21, more preferably 9 to 17 carbon atoms; (B) Polyether-modified siloxane of formula (II) M 1 o D 1 p D′ q Formula (II) Among them, M 1 P 1 3SiO 1 / 2 , D 1 P 1 2SiO 2 / 2 , D′ is P 1 P 2 SiO 2 / 2 , o is 2, p is 0 to 0.1, q is 1.0 to 1.15, P 1 are independently hydrocarbon groups having 1 to 8 carbon atoms, P 2 are independently polyether groups of formula (III), —P 3 O[CH2CH2O] m [CH2CH(CH3)O] n P 5 Formula (III) in m is 3.4 to 11.0, n is 2.5 to 8.0, And the condition is m / n is 1.9 to 2.8, P 3 are independently divalent hydrocarbon groups having 2 to 8 carbon atoms, P 5 is hydrogen; and / or (C) Organic modified polysiloxane of formula (IV) Formula (IV) Where a+b+c+d+2=20 to 210, a is 15 to 205, b is 1 to 12, c is 1 to 12, d is 1 to 12, The R groups are each independently the same or different aliphatic or aromatic hydrocarbon groups having 1 to 10 carbon atoms, R4 groups are each independently the same or different R, R1, R2 or R3 groups, R1, R2 and R3 groups are each independently different polyether groups of formula (V) in e is 3 to 11, f is 6 to 30, g is 0 to 15, h is 0 to 5, i is 0 to 5, and R5 are independently the same or different and are each a methyl group, an acetyl group or a hydrogen group.

12. A method of providing biological protection to a plant or a part thereof, the method comprising contacting the plant or a part thereof with a composition according to any one of claims 1 to 4.

13. Use of a composition according to any one of claims 1 to 4 for providing bioprotection to plants or parts thereof.

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